The detection and identification of cell types in the hematopoietic system has long been a useful research and clinical tool. A number of automated methods exist to aid the researcher and clinician. Among those methods include flow cytometry and fluorescence microscopy. In recent years, the former method has become increasingly sophisticated and has become generally accepted as a tool to aid in the identification of or discrimination between cell types and between various functional and/or maturational subsets within a cell type.
Flow cytometers, which are more generally described in U.S. Pat. Nos. 4,661,913, 4,284,412 and 3,826,364, and in an article by Herzenberg et al., Sci. Am., 234:108 (1976), have been used to identify different populations of leukocytes in a heterogeneous sample by detecting multiple independent parameters on individual cells that pass through the sensing region. Typically, these parameters include forward light scatter (FLS, which is a measure of relative particle size), orthogonal light scatter (OLS, which is a measure of relative granularity) and fluorescence. Fluorescence may be measured from cells that incorporate a nucleic acid or other vital stain or may be measured from cells bearing surface markers which are labelled with monoclonal antibodies (MAbs) that are conjugated directly or indirectly to fluorochromes as described, for example, in U.S. Pat. No. 4,520,110. By combining and comparing these parameters, the various leukocyte components may be distinguished.
U.S. Pat. No. 4,727,020 provides one example of how a flow cytometer operates and may be used to identify leukocyte subpopulations. Unlysed whole blood was treated with one MAb conjugated to phycoerythrin (PE) specific for CD4.sup.+ T cells and and a second MAb conjugated to fluoroscein isothiocyanate (FITC) specific for CD8.sup.+ T cells. A nucleic acid dye, LDS-751.TM. (Exciton), was added to identify nucleated leukocytes. The labelled cells then were analyzed by flow cytometry. A gate was set for LDS-751.sup.+ cells (i.e., for nucleated leukocytes, thereby excluding erythrocytes and platelets). The method allowed separation of leukocyte subpopulations by comparing the various parameters measured.
One problem inherent in any method that makes use of fluorescently labelled MAbs and/or nucleic acid dyes, however, is the propensity of the labels to indiscriminantly bind to damaged cells or cell debris in a sample. This problem is compounded by the fact that in order to prepare cells for labelling, the cells must undergo several preparation techniques, all of which increase the number and proportion of cells that become damaged or ruptured in any one sample. These damaged cells and associated cell debris and their accompanying fluorescence must be discriminated against in the overall sample in order to thoroughly examine the remaining intact cells. Depending upon the method used to prepare the cells in any one sample and even between samples using the same method, sample preparation can introduce a significant amount of variation into the system. As a result, method used for immunofluorescence analysis of cells could mis-identify damaged cells as part of the subpopulations of interest.
A variety of techniques exist for determining whether a cell in a sample is intact or damaged. Viable, intact cells can be distinguished from dead cells by using either fluoroscein diacetate (FDA) or propidium iodide (PI). In these methods, the sample is treated with either FDA or PI and then examined for fluorescence. Cells that stain with FDA are considered "viable"; cells that stain with PI are considered "dead." The methods are limited, however, in that the cells cannot be fixed if the stains are to be used to identify viable cells. Another limitation on the use of FDA is that it brightly fluoresces so that it overwhelms the immunofluorescence signals from other stains, such as FITC and PE, rendering them unreadable.
Other methods to detect intact cells that do not make use of such dyes also exist. FLS, on a flow cytometer for example, may be used to discriminate between intact and damaged cells but only if the cells in the sample are derived from a homogeneous population. Cells from a heterogeneous population cannot be so distinguished because of variations in cell size and light scattering properties.
Each of the above-described methods suffers from some defect that makes it inapplicable as a general method for discrimination between damaged and intact cells in a heterogeneous sample. As a result, there is no single method that allows the researcher or clinician to examine a heterogeneous cell sample from an individual and to discriminate between damaged and intact cells therein.